This invention relates generally to the nucleotide sequences of serogroup-specific capsular polysaccharides genes and their use in a method for typing of serogroups of pathogenic bacteria, in particular Neisseria meningitidis, and further, relates to capsule gene switching in recombinant strains and the detection thereof.
Contagious outbreaks of epidemic diseases constitute public health emergencies requiring rapid treatment and chemoprophylaxis of contacts. Vaccination of the population at risk can be considered if disease cases continue to occur. However, asymptomatic carriage of pathogens in humans is common and some of the adult population may be immunized from previous outbreaks. The factors leading from acquisition of the organism to invasive disease point to a clonal origin of the outbreaks and to an enhanced virulence or altered antigenicity of a particular clone.
Neisseria meningitidis is a leading worldwide cause of meningitis and rapidly fatal sepsis in otherwise health individuals [Apicella, M. A. (1995) in Principles and Practice of Infectious Diseases, eds. Mandell, G. L., Douglas, R. G., and Bennett, J. E., Churchill Livingstone, New York, pp. 1896-1909]. In excess of 350,000 cases of meningococcal disease were estimated to have occurred in 1995 [WHO Report (1996) WHO, Geneva, ISBN 92 4 1561823]. The problem of meningococcal disease is emphasized by the recurrence of major epidemics due to serogroups A, B, and C N. meningitidis over the last 20 years, such as: the devastating serogroup A outbreak in sub-Saharan Africa in 1996 [WHO (1996) Meningitis in Africa. The constant challenge of epidemics. WHO 21:15 March]; the recent dramatic increases in the incidence of serogroup B and C meningococcal disease in parts of North America [CDC (1995) MMWR 44:121-134; Jackson, L. A. et al. (1995) JAMA 273:390-394; Wahlen, C. M. et al. (1995) JAMA 273:383-389]; and the emergence in Europe and elsewhere of meningococci with decreased susceptibility to antibiotics [Campos, J. et al. (1992) J. Infect. Dis. 166:173-177].
Differences in capsular polysaccharide chemical structure determine the meningococcal serogroups [Liu, T. Y. et al. (1971) J. Biol. Chem. 246:2849-58; Liu, T. Y. et al. (1971) J. Biol. Chem. 246:4703-12]. Meningococci of serogroups B, C, Y, and W-135 express capsules composed entirely of polysialic acid or sialic acid linked to glucose or galactose [Liu, T. Y. et al. (1971) J. Biol. Chem. 246:4703-12; Bhattacharjee, A. K. et al. (1976) Can. J. Biochem. 54:1-8], while the capsule of group A N. meningitidis is composed of N-acetyl mannosamine-1-phosphate [Liu, T. Y. et al. (1971) J. Biol. Chem. 246:2849-58]. The currently available capsular polysaccharide vaccines for serogroups A, C, Y, or W-135 N. meningitidis are effective for control of meningococcal outbreaks in older children and adults. However, because of poor immunogenicity in young children and short-lived immunity [Zollinger, W. D. and Moran, E. (1991) Trans. R. Soc. Trop. Med. Hyg. 85:37-43], these vaccines are not routinely used for long-term prevention of meningococcal disease. In the case of group B N. meningitidis, whose (xcex12xe2x86x928)-linked polysialic capsule is an immunotolerized self antigen, a reliable polysaccharide vaccine is not yet available. However, rapid progress is being made in development of polysaccharide-protein conjugate vaccines and it is hoped that following the example of newly licensed Haemophilus influenzae type b vaccines, widespread introduction of the polysaccharide conjugates will lead to elimination of disease.
In some epidemic settings, simultaneous or closely-linked meningococcal outbreaks have occurred in the same population due to different serogroups [Sacchi, C. T. et al. (1994) J. Clin. Microbiol. 32:1783-1787; CDC (1995) MMWR 44:121-134; Krizova, P. and Musilek, M. (1994) Centr. Eur. J. Publ. Hlth 3:189-194]. Further, Caugant et al. (Caugant, D. A. et al. (1986) Proc. Natl. Acad. Sci. USA 83:4927-4931; Caugant, D. A. et al. (1987) J. Bacteriol. 169:2781-2792] and others have noted that meningococcal isolates of different serogroups may be members of the same enzyme type (ET)-5, ET-37 or ET-4 clonal complexes.
Since 1993, the number of cases of serogroup B meningococcal disease in Oregon and adjacent counties in Washington State has doubled, and the overall incidence has been five-fold higher than rates observed in other parts of the United States [CDC (1995) MMWR 44:121-134]. This increase was due to the first appearance in the U.S. of serogroup B meningococcal strains closely related to the ET-5 complex. ET-5 complex strains have been responsible for major epidemics in Norway, Iceland, Cuba and South America over the last twenty years (Caugant, D. A. et al. (1986) Proc. Natl. Acad. Sci. USA 83:4927-4931; Sierra, G. V. et al. (1991) NIPH Annals 14:195-207; Sacchi, C. T. et al. (1992) J. Clin. Microbiol. 30:1734-1738]. Since 1994, cases of serogroup C meningococcal disease due to ET-5 complex strains were also noted in Oregon and Washington State. There exists a recurring need to understand the genetic basis for meningococcal capsule expression and to analyze the serogroup B and C ET-5 meningococcal strains responsible for the outbreak in the Pacific Northwest.
It is an object of the present invention to provide strains of N. meningitidis of a particular serogroup transformed in vitro to express a capsule polysaccharide marker of a different meningococcal strain serogroup. In a particular embodiment are provided prototype serogroup C, Y and W-135 meningococcal strains transformed in vitro with DNA comprising the synD of the serogroup B strain NMB. According to the present invention, conversion from one sialic acid expressing capsule serogroup to another can be accomplished by homologous recombination of the sequences encoding the serogroup-specific capsule polymerase. Such recombinant N. meningitidis strains are provided according to the invention as genetically engineered in vitro recombinations.
Also provided by the present invention are Neisseria meningitidis mutant serogroup strains which express different non-isogeneic capsular polysaccharides due to homologous recombination of the sequences encoding the serogroup-specific capsule polymerase. Specifically exemplified herein is a mutant N. meningitidis strain 1070 (serogroup B, ET-301) in which genetic markers are isogeneic to serogroup B except for the capsule polysaccharide, which is a serogroup C marker. Such meningococcal isolates comprise a recombinant or switched capsule gene and, in a particular embodiment, a switching or recombination event occurred from a serogroup B to a serogroup C capsule biosynthetic gene. Such recombinant N. meningitidis strains are provided according to the invention as naturally-occurring in vivo recombinant isolates.
It is also an object of the invention to provide meningococcal serogroup-specific capsule genes encoding characteristic capsular polysaccharide virulence determinants. In specific embodiments of the invention are provided capsule biosynthetic gene preparations of prototype serogroups A, B, C, Y and W-135, each serogroup-specific gene encoding a biosynthetic enzyme for a specific and distinguishing capsular polysaccharide.
It is an additional object of the invention to provide cloned DNA molecules which can be used to introduce an additional non-isogeneic capsular polysaccharide virulence determinant into strains of N. meningitidis. In a particular embodiment, the cloned DNA fragment containing the stable Tn916 insertion in the synD of the serogroup B N. meningitidis strain NMB was used to introduce the gene for the serogroup B (xcex12xe2x86x928)-linked capsule polysialyltransferase into other meningococcal strains to produce novel immunotypes. More generally, a cloned DNA fragment containing a stable insertion of a polysialyltransferase gene of a specific serogroup strain can be used to introduce the corresponding capsular polysaccharide determinant into serologically different strains to produce novel immunotypes. This invention also contemplates that multiple non-isogeneic capsular polysaccharide virulence determinants can be introduced into serologically different meningococcal strains.
The present invention provides the nucleotide sequence of the intergenic region separating ctrA from the biosynthesis operon (synA-D,E,F,G) of a serogroup A N. meningitidis. Whereas in serogroups B, C, Y and W-135 N. meningitidis, the intergenic region separating ctrA from the biosynthesis operon (synA-D,E,F,G) is 134 bp and contains the ctrA-D promoter as well as the divergent biosynthesis operon promoter and other transcriptional regulatory elements, in serogroup A N. meningitidis the intergenic region is 218 bp in length and does not share any homology with the 134 bp region found in the sialic acid capsular serogroup stains.
This invention also provides evidence that the DNA located between ctrA and galE in serogroup A N. meningitidis is a cassette containing four genes ORF1-ORF4 responsible for the production of serogroup A capsule from UDP-N-acetylglucosamine. Also the organization of, and the amino acid sequences encoded by, the ORF1-ORF4 are provided by the present invention.
Further, according to this invention, the ORF1-ORF4 genes are divergently co-transcribed from overlapping promoters located in a short intergenic region separating the capsule biosynthetic and transport operons. Mutagenesis of these genes results in a capsular phenotype, demonstrating the critical involvement of these genes in serogroup A capsule production.
The invention also provides a model in which meningococcal capsular serogroups are determined by specific biosynthesis genetic cassettes that insert between the ctrA operon and galE. In specific embodiments, it is demonstrated for serogroup A meningococci that the cassettes determining specificity of serogroups can recombine to switch the type of capsule and serogroup expressed. Such information is critical to the design of improved group A and other meningococcal vaccines and to the understanding of the molecular basis of serogroup A pathogenesis.
Also provided are compositions and immunogenic preparations including but not limited to vaccines, as specifically exemplified, comprising at least one capsular polysaccharide derived from one serogroup strain of N. meningitidis and at least one capsular polysaccharide from a different meningococcal serogroup strain, and a suitable carrier therefor are provided. Alternatively, the immunogenic composition can comprise cells of at least two different serotype strains of the specifically exemplified N. meningitidis strains and a suitable carrier.
It is an added object of the present invention to provide protective immunity from virulent meningococcal strains that may not be recognized by traditional serogroup-based surveillance and that can escape vaccine-induced or natural protective immunity by capsule switching. In particular embodiments, the invention provides multivalent vaccines anticipating capsule switching events. According to the invention, broad immunization with capsular polysaccharide vaccines effective against all major capsular serogroups can be used to control epidemics and endemic disease.
It is yet another object of the invention to provide a method for diagnostic detection and serogroup typing of N. meningitidis strains. This method is a nucleic acid amplification (e.g., PCR) method or nucleic acid hybridization method based on (a) the specific nucleotide and encoded amino acid sequences of serogroup-specific capsular polysaccharide determinants and (b) oligonucleotide primers designed to anneal to specific capsule polymerase sequences. This method of the invention was particularly exemplified in the typing of N. meningitidis serogroups A,B,C, Y and W-135. This nucleic acid amplification method of the invention, based on the use of discriminatory primers derived from serogroup-specific nucleotide sequences (Sequo-grouping) offers advantages over current methods of diagnostic detection of serogroup typing in (a) being independent of the need to grow pathogenic organisms for immunological analyses, (b) being capable of being performed directly on clinical specimens, e.g., blood cerebrospinal fluid, with the need to isolate pathogenic organisms, (c) being capable of detecting nucleotide sequences in not only living but also nonliving or nonviable organisms, (d) reducing the exposure of personnel to large volumes of pathogenic bacteria, (e) reducing the cost per serogroup analysis, and (f) improving significantly the accuracy of the serotyping method. This method is particularly preferred as an easy, convenient and rapid screening method for the presence of virulent strains of encapsulated pathogens.